Diabetes mellitus is a metabolic disorder of multiple etiologies, characterized by chronic hyperglycemia with disturbances in carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both. The effects of diabetes mellitus include long-term damage, dysfunction and failure of various organs. Diabetes manifests with characteristic symptoms such as polyuria, polydipsia, weight loss, blurred vision, skin and genital infection [Taft, P., 1984, Pathogenesis, classification presentation, diagnosis and natural/history in diabetes mellitus, A guide to treatment, AIDS Health Science Press. pp: 1-10].
Earlier considered a disease of minor significance to world health, diabetes is taking its place as one of the main threats to human health in the 21st century after cancer and AIDS with an alarming rise in the number of diabetic cases every day. The past two decades have seen an explosive increase in the number of people diagnosed with diabetes worldwide. The global figure of people with diabetes is set to rise from the current estimate of 150 million to 220 million in 2010, and 300 million in 2025 [Amos et al., 1997, Diabetic Med. 14: S1-S85; King et al., 1998, Diabetes Care, 21: 1414-1431]. Recent report released by International Diabetes Federation (IDF) shows that 200 individuals develop diabetes every day [Diabetes voice, a publication of IDF, Nov. 15, 2006]. The Asia Pacific region is at the forefront of the current epidemic of diabetes which constitute 25% of the total 440,000 Type 1 diabetes worldwide under the age of 14 [Cockram, C. S., 2000, Hong Kong Med. J., 6: 43-52]. The Indian scenario is equally alarming, with the age and gender standardized prevalence rate of 4.3% [Sadikot S M, et al; Diabetes India., 2004, Diabetes Res Clin Pract. 66, 301-307]. This when translated into numbers clearly shows that the WHO estimate of the Indian diabetes burden of 35 million people by 2025 [King, H. et al., 1998, Diabetes Care, 21: 1414-1431] has been reached more than two decades earlier. This increase in incidence of diabetes in developing countries is due to the rapid increase in population, increased longevity and high ethnic susceptibility to diabetes coupled with rapid urbanization and lifestyle changes.
Chronic hyperglycemia during diabetes causes glycation of several proteins, thus leading to microvascular complications such as retinopathy, nephropathy, diabetic neuropathy etc. These may be delayed, lessened, or prevented by maintaining blood glucose levels close to normal. Hence, control of blood glucose on a 24 hour basis is the desired goal in the management of diabetes mellitus. Dieting, physical exercise [Bjorntorp, P., 1997, Nutrition 13:795403], insulin replacement therapy, and use of oral hypoglycemic/anti-hyperglycemic agents [Dunn, C. I and Peters; D. H., 1995; Drugs, 49: 721-49] have been employed for the treatment of this disorder, but not with desired success. Although euglycemia can be achieved in diabetic patients by conventional insulin therapy, complications such as hypoglycemia and lipoatropy can not be prevented. Moreover, the cost of insulin treatment is exorbitant, as it is required for the entire lifetime.
Besides the use of insulin, other therapeutic approaches for the control of hyperglycemia include the use of amylin analogues which regulate gastric emptying; inhibitors of intestinal alpha glucosidases like acarbose, miglitol and voglibiose which delay post prandial hyperglycemia; sulphonylureas, the most widely used class of drugs, that act by closure of ATP-dependent IC channel; Metformin, a biguanide that limits intestinal glucose absorption etc. However, these drugs have many adverse effects such as causing severe hypoglycemia at higher doses, liver problems, lactic acidosis and diarrhea [Reuser, A. J. and Wisselaar, H. A., 1994, Eur. J. Clin. Invest., 24(3): 19-24; Dunn and Peters, 1995, Drugs, 49: 721-49; Hillson, R., 1996, Oral hypoglycemic/anti-hyperglycemic treatment, in Practical Diabetes Care, Oxford University Press, pp 62-71].
Since the currently available treatment is expensive and far from satisfactory, alternate therapeutic approaches like the use of medicinal plants for controlling diabetes are gaining popularity among the scientific community.
Scientific investigations have established that M. charantia (bitter gourd) is highly beneficial in the treatment of diabetes in gerbils, langurs, and humans [Baldwa, V. S., et al, 1977, Upsala J. Med. Sci., 82: 39-41; Khanna, P. et al, 1981, J. Nat. Prod., 44: 648-55; Leatherdale, B. A et al, 1981, Br. Med. J. (Clin. Res. Ed.), 282: 1823-1824; Srivastava, Y., et al., 1988, Pharmacol. R. Commun., 20: 201-209]. These investigations strongly suggest the presence of compound(s) with insulinomimetic activities in M. charantia fruits and seeds [Welihinda et al., J. Ethnopharmacol., 17: 277-282, 1986; Srivastava et al., 1993, Phytother. Res. 7: 285-289]. Few attempts have been made to identify the active principles [Khanna, P. et al, (1981) J. Nat. Prod., 44: 648-55; Ng et al., 1986, J. Ethnopharmacol., 15: 107-117].
The published work of Khanna et al., [1981, J. Nat. Prod., 44: 648-55] and their Indian patents (No. 136565 and 176040) describe methods for the extraction of a protein called polypeptide-p from M. charantia fruits and tissue culture. The polypeptide-p is made of 166 amino acid residues and said to be of 11 kDa. The patent describes only the amino acid composition of the polypeptide-p but not the amino acid sequence of the protein. U.S. Pat. No. 6,831,162 describes purification of a hypoglycemic/anti-hyperglycemic protein, polypeptide-k isolated form the dry seeds of M. charantia possessing hypoglycemic/anti-hyperglycemic activity. The polypeptide-k is made up of 160 amino acid residues and is of 18 kDa. Like the Indian patents 136565 and 176040, this patent also gives only amino acid composition and does not disclose the amino acid sequence. Therefore, these molecules are not fully characterized.
Though several attempts have been made by many investigators to obtain a potential hypoglycemic/anti-hyperglycemic drug from M. charantia, it remains an illusion because of several reasons. Lack of systemic investigation, poor statistical analysis, wide variation in preparation technique and optimum dosage of bitter melon [Basch et al., 2003, Am. J. Health Syst. Pharm 60: 356-3591 are responsible for the failures of getting an universal principle from this highly potential medicinal plant. Apart from the above reasons, subtle differences in soil type and its subsequent effect on plant composition result in the variation in the isolated product. Impact of the variation in environmental condition, soil and plant sub-species also play a major role in its protein expression profile [Vallejos, 1991. Plant, Cell and Environment, 14: Page 105; ISB News Report, December, 2004), which influence the potential and reproducibility of the reported data. Production of proteins through recombinant DNA technology allows one to overcome these disadvantages and maintain the quality of the product.
Existing State of Art in Relation to Hypoglycemic/Anti-Hyperglycemic Proteins from M. charantia.                 Indian patent 136565 describes a method for the extraction of a protein called polypeptide-p from M. charantia by using ethanol, diethyl ether and sulfuric acid.        Indian patent 176040 discloses another process claiming it to be more effective than the process described in Indian patent 136565 for the purification of polypeptide-p and uses hexane along with diethyl ether for purification.        U.S. Pat. No. 6,831,162 describes the invention of a novel hypoglycemic/anti-hyperglycemic protein of ˜18 kDa designated as polypeptide-k and its method of extraction.        
These patents though describe the procedure for extraction of hypoglycemic/anti-hyperglycemic proteins from M. charantia; they have several drawbacks:                1. Since the protein is isolated from the plant source, the quality of the isolated product depends on the quality of the raw material used. Since the constituents of the plants are greatly affected by the soil and environment, it is not always possible to get consistent quality of the final product.        2. Seasonal nature of the tropical plant makes it difficult to purify the peptide through out the year. The purification depends upon the availability of the raw material.        3. Extraction of proteins from plant source is time-consuming and cumbersome.        4. Extraction of these polypeptides involves the use of inflammable solvents such as diethyl ether, hexane etc. Use of highly inflammable organic solvents in extraction procedure is not desirable as it exposes the personnel to professional hazards.        5. These solvents are expensive and therefore the extraction is not economical.        6. The recovery of the purified protein is also very poor.        7. The presence of other contaminants in the raw material such as pesticides, insecticides and others affect the quality of the final product.        8. For both the polypeptide-p and polypeptide-k only the number of amino acids present in the protein, amino acid composition and their molecular mass are given. Thus, these proteins are not fully characterized and their primary structure (amino acid sequences) are not defined.        
The present invention discloses a novel hypoglycemic/anti-hyperglycemic protein obtained from the seeds of M. charantia. The present invention further discloses the process for purification of the novel hypoglycemic/anti-hyperglycemic protein of M. charantia and the cloning of the gene encoding the novel hypoglycemic/anti-hyperglycemic protein of M. charantia and expression of this protein in eukaryotic expression system using recombinant DNA technology.